1. Field
The present disclosure relates to intracellular delivery, and relates in particular to compositions for intracellular delivery of therapeutic agents, diagnostic agents, and other materials in the presence or absence of targeting groups. The present disclosure is directed, inter alia, to polymer compositions comprising linear PNAI, cyclic PNAI, linear PEI, and/or cyclic PEI, useful for delivering compounds or substances into a cell. The present disclosure is also directed, inter alia, to methods of using compositions comprising cyclic PNAI and/or cyclic PEI.
2. Description of Related Art
Cells are the basic structural and functional units of all living organisms. All cells contain cytoplasm surrounded by a plasma, or cell, membrane. Most bacterial and plant cells are enclosed in an outer rigid or semi-rigid cell wall. The cells contain DNA which may be arranged in 1) a nuclear membrane or 2) free in cells lacking a nucleus. While the cell membrane is known to contain naturally occurring ion channels, compounds that are therapeutically advantageous to cells are usually too large to pass through the naturally occurring ion channels. Conventional interventional methods for delivering compounds or substances into cells have proved difficult in view of the need for the compounds to pass through the cell membrane, cell wall, and/or nuclear membrane.
Molecular biology has resulted in mapping the genomes of many plants and animals, including the mapping of much of the human genome. The potential for advances in the understanding of the genetic basis of diseases is great, as is the potential for the development of therapies to treat such diseases. To fully take advantage of these advancements and treatment therapies, however, methods are needed for delivering desired compounds into the target cells. Accordingly, researchers developed a variety of intracellular delivery methods for inserting genes and other compounds into both plant and animal cells.
For example, calcium phosphate DNA precipitation has been used to deliver genetic material into cells in cell culture. However, one drawback of this method is that the transfection efficiency (the percentage of transfected cells in a given population) and subsequent gene expression is generally very low.
Improved transfection has been achieved using viral vectors (e.g., adenovirus and retrovirus), but again, difficulties with gene expression have persisted. In addition, substantial concerns regarding antigenicity and the potential of mutant viruses and other possible deleterious effects exist. For example, some viruses may integrate into the genome and facilitate stable expression. If the virus integrates in a way that disrupts normal cell function, however, adverse consequences could result (e.g., cell death, transformation, cancer, etc.).
Liposomes, manufactured more easily than viral vectors, have shown promise as gene delivery agents. Liposomes have fewer biological concerns (for example, they are generally non-antigenic) but the efficiency of transfection and gene expression using liposomes has typically been lower than with viruses.
Gene guns, or biolistic delivery systems, use heavy metal particles (e.g., gold) coated with DNA to fire the particles at high speed into cells. While gene guns have enabled gene expression in culture systems, they have not worked well in vivo. Furthermore, the blast of heavy metal particles may cause damage to the cells and may also introduce undesirable foreign materials, e.g. gold particle fragments, into the cells.
Electroporation is another method of delivering genes into cells. In this technique, pulses of electrical energy are applied to cells to temporarily create pores or openings in the cell to facilitate entry of DNA. Electroporation may damage cells, though, and has not been shown to be highly effective in vivo.
Gene therapy has been heralded as the next revolution in modern medicine, being seen as a potential cure to many diseases both inherited and acquired. Gene therapy is the delivery of genetic information, typically plasmid DNA contained in a vector, to a cell. Typically, the DNA enters the cell via endocytosis and is released into the cytoplasm. Ultimately, the DNA interacts with the host cell environment to (for example) produce proteins encoded by the DNA. One major area of study for gene therapy is the correction of inherited diseases in which a genetic disorder stemming from a malfunctioning endogenous gene may be attenuated by a “healthy” exogenous gene. As a result of extensive genomic research, the genetic makeup of many diseases and their healthy counterparts have been deduced (e.g., cystic fibrosis, Huntington's disease, Alzheimer's disease, and sickle cell anemia), which has spurred on further gene transfer research. The primary obstacle still standing in the way of successful treatment is delivery; it must be cell specific, the gene transfer must be efficient, and the vector must be non-toxic (Putnam, D. “Polymers for Gene Delivery Across Length Scales” Nature Materials Vol. 5 June 2006: 439-451).
The first and most developed area of gene transfer research has utilized viral vectors to introduce DNA. This area has produced some positive results, though the vector itself is inherently flawed. Viruses have evolved the ability to use the host cell's own replication machinery to efficiently and rapidly replicate their own genetic information, which often results in the death of the host cell. To get around this problem, viruses used for transfection are genetically modified to be replication defective. This requires the removal of its virulent genetic information and the insertion of a therapeutic gene. The initial results from early clinical trials using this technique were positive, but early success was soon diminished when three cases of leukemia-like complications were detected in participants of a clinical trial (Wong, S. Y., J. M. Pelet, D. Putnam. “Polymer systems for gene delivery—Past, Present, and Future” Progress in Polymer Science Vol. 32 April. 2007: 99-837). The virus's random transgenic insertion of its genetic payload into the host cell chromosome was to blame, since it could potentially insert into an area that coded for a protein responsible for the regulation of cell growth and division. Other potentially lethal complications that may occur using a viral vector include initiation of an immunological response by the host, as well as the potential for the vector to travel to disease-free tissue.
The clarification and correction of these complications has become a major area of interest in this field. At the same time many have turned to non-viral delivery systems to find a safer method of gene delivery, including delivery of naked DNA by physical methods, lipid based vectors, and synthetic polymer vectors (Taira, K., K. Kataoka, T. Niidome. Non-viral Gent therapy: Gene Design and Delivery. Tokyo, New York Springer Science & Business Media, 2005). Delivery of free plasmid DNA via electoporation into a cell has been an enticing approach, given the absence of an immune response that is more evident in molecular vector systems. Electroporated DNA is induced to enter a cell by an application of electric or magnetic fields to the targeted tissue, which increases the permeability of cell membranes. Although this is one of the most precise methods to target a certain tissue, it is not cell specific and requires high levels of unencapsulated DNA, which has been shown to lead to high blood pressure and slow heart rates (Taira, K, 2005). An alternative method is to form hydrophobic lipoplexes, liposomes that associate with DNA, which are more readily taken up through interactions with the cell's phospholipid bilayer. Combined with the addition of a ligand or signaling sequence, these vectors can be more efficient at entering targeted cells.
Payload as well as transfection efficiency have been shown to increase when lipid based delivery is used in conjunction with cationic polymers (Wong, S. Y., 2007). Charged polymers, such as polyethylenimine (PEI), have been incorporated into vector systems called polyplexes, which have become popular because of their ability to be manipulated in the laboratory to achieve desired characteristics; however some obstacles still stand in the way. A current challenge in the design of cationic vectors is overcoming cytotoxicity. A number of researchers have studied the effects of adding further modifications to enhance biocompatibility. The exact mechanism that causes cytotoxicity is not entirely certain, but the leading hypothesis is that ionic interactions between the cationic moieties of the vector and the anionic domains on the cell surface lead to polyplex aggregation on the outer plasma membrane (Wong, S. Y., 2007). The cytotoxic effect has been shown to be caused and exacerbated by several physical properties including molecular weight (MW), degree of branching, charge density, cationic functionality type, three dimensional conformation, as well as polyplex size, surface area and flexibility (Wong, S. Y., 2007). Of the different properties that increase toxicity, MW has been shown to be one of the leading parameters. This has posed a crucial dilemma, since increasing the MW within a certain limit is also beneficial to transfection efficiency (Wong, S. Y., 2007). Other problems that arise when using cationic vectors include introducing DNA into non-target cells, and the systemic stability of the polyplex in the blood stream.
The present disclosure provides new and/or better methods for delivering compounds, including genetic material, into a cell. The methods of the present disclosure provide a significant advantage over prior art methodology in that enhanced levels of intracellular delivery and—in the case of nucleotides—gene expression may be achieved. In addition, the methods of the present disclosure may be performed in cell lines which may be otherwise resistant to intracellular delivery and gene expression using other conventional means. These and/or other aspects of the present disclosure will become apparent from the further discussions herein.